WO1999027139A1 - Method and apparatus for preserving human saliva for testing - Google Patents
Method and apparatus for preserving human saliva for testing Download PDFInfo
- Publication number
- WO1999027139A1 WO1999027139A1 PCT/US1998/025089 US9825089W WO9927139A1 WO 1999027139 A1 WO1999027139 A1 WO 1999027139A1 US 9825089 W US9825089 W US 9825089W WO 9927139 A1 WO9927139 A1 WO 9927139A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- kit
- solution
- metabolites
- enzyme
- hydrophilic compound
- Prior art date
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B10/0045—Devices for taking samples of body liquids
- A61B10/0051—Devices for taking samples of body liquids for taking saliva or sputum samples
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/16—Reagents, handling or storing thereof
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0633—Valves, specific forms thereof with moving parts
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0633—Valves, specific forms thereof with moving parts
- B01L2400/0672—Swellable plugs
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
- B01L2400/0683—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers mechanically breaking a wall or membrane within a channel or chamber
Definitions
- This invention relates to a method for the preservation of bodily fluid samples. More particularly, this invention relates to a method for the preservation and storage of human saliva samples for use in subsequent drug testing.
- the present manner of testing therefore results in at least two problems: 1) the employee is required to leave their job to undergo testing when they could otherwise be working; and 2) the privacy required by urine tests affords the employee the opportunity to submit a fraudulent sample (e.g., the employee could obtain a sample from another person and submit that drug free sample for testing).
- an employer would be desirable for an employer to have a method of testing an employee at their place of business, with only a minimum level of personal inconvenience to the employee.
- a similar method is useful in assisting a law enforcement officer preserve a suspect saliva sample for later forensic testing, e.g., to determine an individual's blood alcohol or other drug level.
- a qualitative test could be performed at the time of saliva collection to determine if quantitative testing is justified.
- the present invention to provides a method of preserving saliva in a liquid solution for subsequent chemical assays. This method would allow the employee to remain at his or her place of business, require only a minor inconvenience during testing and provide the necessary safeguards against submission of fraudulent tests while insuring that an accurate and precise drug test can be taken at a later time.
- the present invention is also useful in providing a qualitative, instantaneous test for a drug after the saliva sample has been tamper-proof sealed.
- Figure 1 is a perspective view of a specimen cup of the instant invention
- Figure 2 is a cross-sectional view of a capillary of the specimen cup shown in Figure 1.
- the present invention is a method and composition for the preservation of a saliva sample for use in subsequent quantitative chemical assays.
- the method involves collecting a saliva sample at a location, directly into a specimen cup.
- the specimen cup containing a predetermined volume of aqueous solution of pH buffered saline and an enzymatic inhibitor.
- the specimen bottle is optionally adapted with a constituent compound specific, qualitative test unit contained within a capillary extending from the bottle.
- the qualitative test unit contains vacuum packed ampoule containing dried enzyme, a solution swellable plug and a suitable colorometric reagent. Upon breaking the ampoule, a predetermined volume of solution is drawn into the capillary, thereby activating the qualitative test unit. The solution swellable plug shortly thereafter swells to close off the qualitative test volume from the specimen cup contents. After a tamper-proof seal has been made the specimen cup is then transported to an off-site second location, where the saliva sample-solution is quantitatively assayed for a saliva constituent compound by conventional means common to blood or urine assays.
- hydrophilic compounds in general and alcohol in particular, once absorbed from the intestinal tract, and into the bloodstream are evenly mixed into the total body water of the body.
- a hydrophilic compound is defined as a substance that is found in the body plasma, either in the administered form or as a metabolite thereof. While the description details a method and composition for the preservation of a saliva sample for determination of ethanol content, it is appreciated that the instant invention is operative for the measurement of various other hydrophilic compounds absorbed and excreted by the parenchyma.
- Fat tissues include tissues or tissue fractions bounded by lipid membranes such as erythrocytes. Hydrophilic compounds enter such a tissue, but are not dissolved into the fat, but rather into the water contained within that tissue.
- alcohol for example, is entirely found after several circulation times to be in a volume of approximately 0.60-0.68 liters/kg in a male, and about 0.52-0.54 liter/kg in a female. Once into the body water, alcohol is distributed throughout this volume of water and is subjected to metabolism, excretion, partitioning and excretion limits.
- Some parts of blood are essential for the perfusion of glandular tissues such as the exocrine glands of the alimentary tract - those glands of the mouth and buccal cavity, the pancreas and other organs lower in this path.
- glandular tissues such as the exocrine glands of the alimentary tract - those glands of the mouth and buccal cavity, the pancreas and other organs lower in this path.
- the perfusion of the salivary glands of the pharyngeal and buccal cavity including the parotid glands, the submaxillary glands, and the sub lingual glands are of importance to this method.
- nutrients amino acids, carbohydrates and fats
- bulk water are taken from the capillary bed(s) of these glands, and are exposed to the individual cells of the gland.
- Such cells are commonly called the "parenchymal” cells of the gland - e.g., the cells that "secrete” water, protein or other substances (mucin, etc.). It is the “bulk water” fraction, e.g., the water present in the parenchyma, that composes the fluid portion of any secretion from a gland. Finally, a substance dissolved within the “bulk water” of the gland, is often excreted when the gland is called upon to excrete. In the case of any of the salivary glands, water, and some protein material is excreted into the saliva. Thus, excretions of the salivary glands are composed of an isotonic or slightly hypertonic aqueous salt solution, generated from blood plasma.
- excretions can also contain various enzymes as are characteristic to the gland, the various enzymes having proteolytic activity to break down or metabolize proteins to peptides and/or amino acids; complex carbohydrate cleavage properties; and to a lesser extent lipid metabolizing properties.
- Ethyl alcohol when present in the plasma (or blood) from the consumption of ethanol, is a component of the blood that perfuses the salivary glands. It is known that alcohol is extracted into the saliva and that it is concentrated from the plasma during this process, so that, in humans, there is a concentration of 8-15% over the concentration present in an equivalent blood sample. Saliva ethanol content has been measured to be about 9% higher than in capillary blood, C. Lenter, Geigy Scientific Tables, Vol. 1, Units of Measurement, Body Fluids, Compositions of the Body, Nutrition, Basle: Ciba-Geigy, 1981; which is incorporated herein by reference. In a saliva sample, measurement of blood alcohol level is determined by quantifying the alcohol concentration in a saliva sample.
- any random "spit" of saliva from the mouth will average approximately 2.0 milliliters, typically ranging from 1.85 to about 2.35 milliliters.
- Such a sample of saliva can be used for the estimation of the concentration of alcohol present in the blood that perfused the salivary glands producing the saliva sample.
- substances are optionally contacted with the buccal cavity to generate a reflex stimulation of saliva by the above named glands.
- these substances illustratively include citric acid (e.g., a lemon wedge) or milk.
- the sample of saliva when caught and preserved in a suitable solution is subsequently used to estimate a blood concentration of alcohol in the person from whom it is taken.
- the solution into which the saliva is placed includes an agent for lessening the degradation of the saliva by the inhibition of enzymatic metabolism of the alcohol or test substance present in the sample by bacteria, fungi, white blood cells, macrophages, or other organisms that can reside in the environment of the buccal or respiratory cavities of the sample donor.
- the solution contains an ionic solute present at a concentration in the range of osmalities associated with normal physiological body fluids.
- the body fluids including body plasma, urine and saliva.
- a specimen solution is prepared which contains a salt and dilute aqueous protein matrix solution that mimics body plasma or urine, and an enzymatic inhibiting agent.
- a salt and dilute protein matrix solution that mimics body plasma or urine, and an enzymatic inhibiting agent.
- Commercially available salt and dilute protein matrices are operative in the instant invention.
- a salt and protein matrix mimics the osmality, composition, pH and general properties of body plasma or urine.
- the known parameters associated with body plasma serves as a baseline calibration for quantitative analysis of the sample within the matrix solution.
- the constituent substances of such a salt and protein matrix illustratively includes: bicarbonate, calcium, chloride, phosphate, potassium, sodium, sulfate, sulfite, albumins, amino acids, nucleotides, nucleosides, urea, creatine, citrate, formate and lactate.
- the salt and protein matrix is optionally replaced by a solution conventionally used for the storage of bodily fluids, illustratively including: a buffered salt solution of isotonic saline (0.085 g/L NaCl); and 50 mM phosphate buffered saline.
- the pH of such a solution is preferably between 7 and 8.
- An enzymatic inhibiting agent of the instant invention is present in a concentration from 0.01 to 10 mole percent, relative to the specimen solution water.
- the agent serves to arrest the action of enzymes that degrade substances such as drugs or alcohol within living cells contained in the sample or in the solution of the specimen cup.
- the inhibition of alcohol dehydrogenase is of particular concern.
- the enzyme inhibiting agent is present from 0.05 to 1 mole percent relative to the specimen solution.
- Representative enzymatic inhibiting agents of the instant invention include: aminoglycosides, cephelosporins, tetracyclines, sulfa-drugs, penicillins and similar antibiotics.
- the optimal enzymatic inhibiting agent concentration is dictated by the efficacy of the specific compound in disrupting enzymatic activity.
- the agents of the instant invention also may have secondary biocidal effects on organisms present in the specimen cup.
- the enzymatic inhibiter functions to interfere with glycolysis pathway reactions.
- a fungicide or mycocide is added to the specimen solution.
- the fungicide (or mycocide) is present in a concentration from about 0.01 to 10 mole percent, relative to the specimen solution water. More preferably, the fungicide (or mycocide) is present in a concentration from about 0.05 to 1 mole percent, relative to the specimen solution water.
- Fungicides or mycocides operative in the instant invention illustratively include: polymyxins, polynoxylins, nystatin, hedaquinium chlorides, tetrachloroisophtalonitrile and ketoconazole.
- a bactericide is added to the specimen solution.
- the bactericide is present in a concentration from about 0.01 to 10 mole percent, relative to the specimen solution water. More preferably, the bactericide is present in a concentration from about 0.05 mole percent, relative to the specimen solution water.
- Bactericides operative in the instant invention illustratively includes: aninoglycosides, cephelosporins, tetracyclenes, sulfa-drugs, penicillins and similar antibiotics. The order by which these reagents are prepared or mixed is not essential and has no bearing on the ultimate utility of the solution in the instant invention.
- the above reagents should be well mixed, and preferably dispensed into sterile containers, with a volume of between 10 and 40 milliliters and preferably of at least 15 and less than about 18 ml.
- This volume of material and reagent is well suited to analysis by a laboratory to determine the concentration of alcohol present in the specimen cup by conventional techniques such as an alcohol dehydrogenase assay.
- a different volume of specimen cup solution is utilized for collection of a sample, as analysis techniques dictate. It is appreciated that dilution of the sample with large volumes of specimen cup solution may require a primary amplification of the sample to produce accurate assay results.
- the specimen cup preferably has means of sealing so as to prevent tampering or opening prior to testing.
- the specimen cup more preferably has a port for the extraction of a test aliquot.
- a test participant In the operation of the instant invention, a test participant expectorates into a specimen cup containing matrix solution. Once a sample has been taken, the cup is sealed and transported to an off-site laboratory for later testing. Preferably the sample containing specimen cup is stored at a temperature between about 4°C and 25°C.
- mg/dl milligrams per deciliters
- a second embodiment of the instant invention applicable to blood alcohol testing inco ⁇ orates a means for quantitating the amount of alcohol present in the sample at the sample gathering location, or by having a reagent system that performs the required chemistry, and a coupled detector system that allows analysis and/or visualization of the sample.
- an in situ analysis solution additionally contains reagents for performing analysis of alcohol using an enzymatic assay inco ⁇ orating the formation of NADPH from NADP acting as a cofactor in conjunction with the enzyme alcohol dehydrogenase conversion of ethanol to acetaldehyde.
- alcohol oxidase alone or in concert with a peroxidase enzyme, also may provide a colorometric redox product.
- alcohol in the saliva sample reacts with the enzyme and excess NADP to form a product, acetaldehyde, and the reduced cofactor, NADPH in quantitative yield.
- these enzymes are known to the art, as is the usage of catalytic antibodies for performing redox chemistry on constituent compounds.
- a further reagent required to determine the concentration of alcohol from a saliva sample causes an interaction of NADPH with nitro blue tetrazolium
- NBT NBT will interact with NADPH in a quantitative manner to form a reduced formazan. Such compounds are intensely colored - usually dark blue, and are thus readily quantitated by spectrophotometric means.
- Figure 1 shows a specimen cup generically at 10 designed for on the spot qualitative detection of a saliva constituent compound and subsequent quantitative assays.
- a threaded lid 12 is adapted to selectively seal the mouth 14 of a bottle 16.
- a tamper-proof adhesive tape is optionally deployed in contact with both the lid 12 and the bottle 16 following the collection of a saliva sample (not shown).
- the bottle 16 has at least one hollow capillary 18 extending from the bottle wall 20.
- the capillary 18 extends from the bottle wall 20 at a position so as to assure that the specimen solution within the bottle 16 covers the capillary opening when the specimen cup 10 is disposed in an upright position.
- the capillary 18 is in integral part of the bottle 16.
- the bottle 16 being injection molded of a suitable thermoplastic material.
- the bottle 16 is of a clear or translucent appearance.
- a specimen cup designed for on the spot qualitative detection of a saliva constituent compound is designed to withdraw a predetermined solution volume for detection and then to isolate that volume from the bulk of the specimen solution. Isolation of the detection volume assures that the reagents of the qualitative detection do not interfere with the subsequent qualitative assays.
- Figure 2 shows a cross-sectional view of the capillary 18.
- the wall thickness 22 of the capillary 18 is optionally less than that of the bottle wall 20.
- a thin capillary wall is flexible and allows the capillary to be bent.
- a thin walled ampoule 24 is adapted to insert within the bore of the capillary 18. The ampoule 24 fills the majority of the capillary bore volume.
- the ampoule 24 contains the reagents for in situ colorometric detection collectively shown at 26, as well as a solution swellable plug material 28.
- the reagents 26 include a freeze dried enzyme specific to the saliva constituent of interest and a suitable redox activated colorometric indicator.
- the enzyme and indicator are mixed with an inert substrate such as glass wool.
- the solution swellable plug 28 is preferably an inert hydrophilic polymer which is susceptible to rapid hydration upon contact with the specimen solution.
- the solution swellable plug 28 is illustratively cellulose, carboxymethyl cellulose, gelatine, alginates, and mixtures thereof.
- An ampoule containing the constituent compound specific reagents and a swellable plug material is inserted into the flexible capillary.
- a measured amount of solution is sealed in the bottle.
- a saliva sample is collected by the user removing the lid and expectorating into the bottle, thereafter the bottle is resealed and optionally tamper-proof sealed with an adhesive tape.
- the capillary is bent so as to break the ampoule contained therein.
- solution fills the capillary in order to equilibrate the pressure between the capillary and the head space within the specimen cup bottle.
- the amount of solution drawn into the capillary is controlled by the pressure and volume of the ampoule.
- an ampoule of the instant invention is constructed from a glass tube, such as a Pasteur pipette or melting point tube.
- One end of the glass tube is flame sealed and then the swellable plug material and the detection reagents sequentially added to the tube.
- the open end of the ampoule then engages a vacuum line in order to reduce the pressure within the vial. While the vacuum line is evacuating the tube, a region of the tube above the reagents is softened by means of a heat source until the tube seals and is drawn free of the vacuum line.
- the vacuum line is maintained by a conventional means such as a mechanical rotary pump or an aspirator.
- the vacuum sealed ampoule is optionally scored or otherwise weakened at a specific point in order to facilitate a controlled fracture.
- the following compounds are individually operative as enzymatic inhibitor components of a specimen solution of the instant invention.
- the approximate efficacious concentrations for individual enzymatic inhibitor components is also provided.
- Example 3 The method as described in Example 3 is repeated with an aliquot being assayed by conventional gas chromatography techniques for methylamphetamine.
- Example 3 The method as described in Example 3 is repeated with an aliquot being assayed by conventional gas chromatography techniques for catecholanine. An error of between 5 and 9 concentration percent is observed for the saliva-test based levels of catecholanine obtained from the instant invention, as compared to the test subject blood.
- Example 3 The method as described in Example 3 is repeated with an aliquot being assayed by conventional gas chromatography techniques for an opiate. An error of between 6 and 12 concentration percent is observed for the saliva-test based levels of an opiate or opiate metabolite obtained from the instant invention, as compared to the test subject blood.
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000522280A JP2001524321A (en) | 1997-11-26 | 1998-11-24 | Method and apparatus for storing human saliva for testing |
NZ505342A NZ505342A (en) | 1997-11-26 | 1998-11-24 | Method and apparatus for collecting and preserving human saliva for drug and alcohol testing |
CA002310616A CA2310616C (en) | 1997-11-26 | 1998-11-24 | Method and apparatus for preserving human saliva for testing |
AU16021/99A AU754598B2 (en) | 1997-11-26 | 1998-11-24 | Method and apparatus for preserving human saliva for testing |
DE69839613T DE69839613D1 (en) | 1997-11-26 | 1998-11-24 | METHOD AND APPARATUS FOR PRESERVING STORE FOR ANALYZES |
EP98960428A EP1032709B1 (en) | 1997-11-26 | 1998-11-24 | Method and apparatus for preserving human saliva for testing |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/978,729 US5968746A (en) | 1997-11-26 | 1997-11-26 | Method and apparatus for preserving human saliva for testing |
US08/978,729 | 1997-11-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999027139A1 true WO1999027139A1 (en) | 1999-06-03 |
Family
ID=25526342
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1998/025089 WO1999027139A1 (en) | 1997-11-26 | 1998-11-24 | Method and apparatus for preserving human saliva for testing |
Country Status (9)
Country | Link |
---|---|
US (2) | US5968746A (en) |
EP (1) | EP1032709B1 (en) |
JP (1) | JP2001524321A (en) |
AT (1) | ATE397891T1 (en) |
AU (1) | AU754598B2 (en) |
CA (1) | CA2310616C (en) |
DE (1) | DE69839613D1 (en) |
NZ (1) | NZ505342A (en) |
WO (1) | WO1999027139A1 (en) |
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US11002646B2 (en) | 2011-06-19 | 2021-05-11 | DNA Genotek, Inc. | Devices, solutions and methods for sample collection |
US11536632B2 (en) | 2011-06-19 | 2022-12-27 | DNA Genotek, Inc. | Biological collection system |
US11549870B2 (en) | 2011-06-19 | 2023-01-10 | DNA Genotek, Inc. | Cell preserving solution |
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Also Published As
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EP1032709A4 (en) | 2007-01-10 |
DE69839613D1 (en) | 2008-07-24 |
AU1602199A (en) | 1999-06-15 |
US6291178B1 (en) | 2001-09-18 |
CA2310616C (en) | 2009-02-03 |
JP2001524321A (en) | 2001-12-04 |
NZ505342A (en) | 2002-03-28 |
US5968746A (en) | 1999-10-19 |
EP1032709B1 (en) | 2008-06-11 |
CA2310616A1 (en) | 1999-06-03 |
EP1032709A1 (en) | 2000-09-06 |
AU754598B2 (en) | 2002-11-21 |
ATE397891T1 (en) | 2008-07-15 |
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